Epigenetic modifications of histone and DNA control gene expression and critically shape phenotypes and cell states. These modifications are tightly controlled by interactions with a constellation of trans-acting regulatory factors, and are dysregulated in a plethora of diseases. Next-generation sequencing (NGS) technologies have allowed genome-wide profiling of these modifications in diverse cell types, normal and disease conditions as well as across individuals. However, these strategies have yielded mostly associative and/or correlative insight into relationships between epigenetic state, gene expression and phenotype with limited power to establish the causality of individual epigenetic modifications that is fundamental to understanding normal physiology and diseases. Existing techniques to investigate phenotypic consequences of epigenetic gene regulation irreversibly delete stretches of genomic sequence that can have multiple functions, or perturb protein factors that control thousands of genes, confounding accurate conclusion. A scalable toolbox allowing in situ and in vivo combinatorial modifications of epigenetic states would offer unprecedented opportunities for both mechanistic studies and unbiased discovery of novel functional elements at the genomic scale. This project will respond to this need by developing an expandable molecular toolkit to precisely and reversibly manipulate defined epigenetic modifications (e.g., H3K27ac) at defined genomic addresses based on our innovative CRISPR/Casilio platform. There are three Specific Aims in this project. Aim 1 is focused on the development of a comprehensive set of epigenetic editing modules with which Casilio can achieve multiplexed and combinatorial edits, whereby different epigenetic modifications can be elicited simultaneously at distinct genomic loci while multiple modifications can also be induced in each locus. Aim 2 will deliver Casilio-enabled cell lines with which scientists can achieve epigenome editing with unprecedented ease by delivering short RNA guides. Aim 3 will center on the development of genome-wide guide libraries targeting genomic regulatory elements such as enhancers and insulators with which scientists can perform reverse epigenetic screens to discover novel elements and epigenetic modifications causative to phenotype. This toolkit, once developed, will transform the ways we study epigenetics by providing a fine and scalable technique to directly edit epigenetic states at defined targets, to investigate the underlying causes of gene regulatory changes observed in biological processes and diseases. To truly benefit the field of functional genomics, we will share our methodology and reagents at every stage of platform development with the scientific community. Through the establishment of standards and a module registry as well as reagent sharing via an open repository, we hope to create a sustainable ecosystem of Casilio module users and developers to apply and expand the Casilio toolbox for epigenetic editing. Due to the programmability and precision, epigenetic editing enabled by Casilio may provide new means and tools for powering personalized and precision medicine.